Activity modulation of anti-sigma factor via cysteine alkylation in Actinobacteria

σ^R (SigR) is an alternative sigma factor that enables gene expression in Streptomyces coelicolor to cope with thiol oxidation and antibiotic stresses. Its activity is repressed by a zinc-containing anti-sigma (ZAS) factor RsrA that senses thiol oxidants and electrophiles. Inactivation of RsrA by disulfide formation has been well studied. Here we investigated another pathway of RsrA inactivation by electrophiles. Mass spectrometry revealed alkylation of RsrA in vivo by N-ethylmaleimide (NEM) at C61 and C62 located in the C-terminal loop. Substitution mutation (C61S/C62S) in RsrA decreased the induction of σ^R target genes by electrophiles and made cells more sensitive to electrophiles. In contrast to stable protein of oxidized RsrA, alkylated RsrA is subjected to degradation partly mediated by ClpP proteases. RsrA2, a redox-sensitive homolog of RsrA in S. coelicolor lacking cysteine in the terminal loop, did not respond to electrophiles. However, redox-sensitive RsrA homologs in other Actinobacteria also harboring terminal loop cysteines all responded to electrophiles. These results indicate that the activity of RsrA can be modulated via cysteine alkylation, apart from disulfide formation of zinc-coordinating cysteines. This pathway expands the spectrum of signals that the σ^R -RsrA system can sense and reveals another intricate regulatory layer for optimal survival of Actinobacteria.